Studies of the intermediates in the biosynthesis of myoinositol from glucose 6-phosphate have been extended. Two enzymes, L-myoincsitol 1-phosphate synthase (EC 5.5.1.4) and myoinositol 1-phosphatase (myoinositol 1-phosphate phosphohydrolase, EC 3.1.3.25) acting sequentially isomerize glucose 6-phosphate to myoinositol 1-phosphate by an NAD+ dependent oxidoreduction followed by Mg2+ dependent hydrolysis to free myoinositol, the pathway de novo of cyclitol biosynthesis. The synthase has been purified to homogeneity and the phosphatase has been purified 40-fold. The requirement for NAD+ in an isomerization reaction implies the intermediate formation of oxidized species which are ultimately reduced by NADH generated concomitantly. An intramolecular aldol condensation is the likely mechanism of cyclization. The postulated model then is oxidation of glucose 6-phosphate to 5-ketoglucose 6-phosphate, cyclization to inosose 1-phosphate, and reduction by NADH to L-myoinositol 1-phosphate, the two intermediates bound tightly to the enzyme and undetectable by any direct method. Through interruption of the normal enzymatic reaction with NaB3H4 both ketonic intermediates should be reduced to pairs of labeled epimeric alcohols. After dephosphorylation the addition of the expected reduced products as carriers followed by re-isolation will, if labeled, reveal the presence of the intermediates. Of the four carriers added, iditol, glucitol, scyllitol, and myoinositol, only the latter two were labeled. Since they are uniquely related to inosose, inosose 1-phosphate is confirmed as an intermediate, the amount of label incorporated supporting the formation of 1 mole of inosose 1-phosphate per mole of enzyme. The alditols were unlabeled in the epimeric 5-positions implying that 5-ketoglucose 6-phosphate is extremely short-lived and further that both the oxidation and cyclization are fast reactions. H isotope effects observed earlier in this reaction can now be localized to the reductive step which must be the slow or rate-limiting reaction. The phosphatase is equally active toward both optical isomers of myoinositol 1-phosphate, suggesting an additional crucial role for the enzyme in the recycling of myoinositol through phosphatidylinositol.